The rodent eye as a non-invasive window for understanding cancer nanotherapeutics Abstract: We propose to use the mouse eye as a non-surgical window for highly efficient, optical investigation of subretinal xenograft models and nanodelivery, using a state-of-the-art ocular imager, EyePod. The EyePod employs single-cell resolution intravital confocal microscopy and optical coherence tomography, performed completely non-invasively through the natural optics of the eye. This technology enables repeatable in vivo imaging over weeks and even months, quantitative tracking of tumor development and delivery of theranostic nanoparticles, and the measurement of tumor and tissue responses. The novel micellar-based nanoporphyrin that we have recently reported will be used in this project. This exciting and highly versatile theranostic porphyrin/cholic acid-based micellar nanoparticle allows (i) efficient encapsulation of hydrophobic chemotherapeutic drugs or fluorescent dyes, (ii) near- infra red fluorescent (NIRF) detection of the tumor via the intrinsc fluorescence of porphyrins, (iii) photodynamic therapy (PDT) and photothermal therapy (PTT) via efficient free radical and heat generation at the tumor site, respectively, (iv) Gd(III) loadin for MRI imaging, (v) 64Cu loading for PET imaging, and (vi) convenient ligation of cancer-targeting ligands to the surface of the micelle for cancer- specific targeted delivery. Thorough understanding of how this nanocarrier distributes within the tumor microenvironment, and how it responds to controlled optical stimulation will enable us to maximize its therapeutic potential as a nano-theranostic agent. We have also recently developed a short peptide that is specifically taken up by mouse brain vascular endothelial cells. Specific Aims are: 1. To develop intraocular glioblastoma and breast cancer xenograft models in eyes of nude mice, and to use non-invasive optical techniques (the EyePod) to study the development of these tumor models at cellular resolution longitudinally over days and weeks, and their response to treatment with nanodoxorubicin. 2. To use the tumor models and EyePod to study the biodistribution and photo-response of tumor targeting and non-targeting nanoporphyrins within the tumor micro-environment in vivo, and to use cryo-electron microscopy to dissect the nanodelivery at the ultrastructural level. 3. To optimize brain vascular endothelial cell-penetrating ligands, and to use in vivo EyePod imaging and cryo-electron microscopy to study their nanodelivery into retinal vasculature, and across the blood retinal barrier, which is very similar to blood brain barrier. Hypothesis & Impact: We hypothesize that the mouse eye provides a unique and convenient window to study: (i) tumor development, (ii) biodistribution of nanoparticle drugs or imaging agents inside the tumor microenvironment, and (iii) biology of tumor responses to the nanotherapeutics. By combining advanced optics with state-of-the art label-free and fluorescence labeling, we will be able to examine at micrometer resolution the location and fate of the nanoparticles and the encapsulated drug in real time over days and weeks. Information gained from this study will not only facilitate understanding of nanoparticle penetration into the tumor vasculature, distribution inside the tumor stroma, and uptake into tumor cells, but will also allow us to establish robust protocols for efficient study of tumor development, tumor microenvironment, in vivo intra-tumoral biodistribution and fate of drugs encapsulated by nanocarrier, and nanodelivery across the BRB.